The presence of nucleic acids in samples has traditionally been determined using Southern blot analyses. However, these methods are limited in typically requiring denaturation of the nucleic acids. Moreover, the old art methods are typically not well adapted to quantitation of the nucleic acids, detection of double stranded nucleic acids versus single stranded nucleic acids, or detection of samples in complex environments, such as tissue samples.
Detection and quantitation of RNA has traditionally been measured using Northern blot, dot blot, and nuclease protection assays. However, these approaches are time-consuming, have limited sensitivity, and the data generated are more qualitative than quantitative in nature. Greater sensitivity and quantification is often possible with polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) based methods, such as quantitative real-time RT-PCR, but these approaches have low multiplex capabilities (Bustin (2002) “Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems” J Mol Endocrinol 29:23-39 and Bustin and Nolan (2004) “Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction” J Biomol Tech. 15:155-66).
Microarray technology has been widely used in discovery research, but its moderate sensitivity and its relatively long experimental procedure have limited its use in high throughput expression profiling applications (Epstein and Butow (2000) “Microarray technology—enhanced versatility, persistent challenge” Curr Opin Biotechnol. 11:36-41).
Other problems not well addressed in the art are detection of hybridized nucleic acid pairs, localization of certain nucleic acids in complex samples, and simultaneous detection of DNA and mRNA in the same sample.
In Kennedy (J. Histochem & Cytochem 50(9): 1219-1227, 2002; and U.S. Pat. No. 7,033,758), HPV DNAs were detected by in situ hybridizations, e.g., using bDNA technology. The method requires RNAse digestion to avoid possible background and false positive signals from sample RNA sequences. The method was able to distinguish different HPV subtypes and localize the position of the target DNA within a cell. However, the methods were unable to detect RNA or confirm the double stranded status of the nucleic acids.
In Byrom (Ambion Technotes 9(3); Dec. 6, 2004), double labeled, double stranded siRNAs were used to inhibit expression of certain proteins. The siRNAs were stable enough in live host cells to be followed through the course of cell divisions. In one experiment, the anti-sense siRNA strand against c-myc included a red fluorescent reporter, and the sense strand a green fluorescent reporter. Under examination by fluorescence microscope, the double stranded siRNA appeared yellow, e.g., as it was transforming HeLa cells in a cationic lipid carrier. Separated single probe strands were followed, but without any information of their hybridization state.
In view of the above, a need exists for ways to determine the location of certain nucleic acids, and their single or double stranded character. It would be desirable to have systems that can distinguish between DNA and RNA in a cell or tissue sample. The present invention provides these and other features that will be apparent upon review of the following.